Whole-Transcriptome and -Genome Analysis of Extensively Drug-Resistant Mycobacterium tuberculosis Clinical Isolates Identifies Downregulation of as a Mechanism of Ethionamide Resistance.

Genetics-based drug susceptibility testing has improved the diagnosis of drug-resistant tuberculosis but is limited by our lack of knowledge of all resistance mechanisms. Next-generation sequencing has assisted in identifying the principal genetic mechanisms of resistance for many drugs, but a significant proportion of phenotypic drug resistance is unexplained genetically. Few studies have formally compared the transcriptomes of susceptible and resistant strains. We carried out comparative whole-genome transcriptomics of extensively drug-resistant (XDR) clinical isolates using RNA sequencing (RNA-seq) to find novel transcription-mediated mechanisms of resistance. We identified a promoter mutation (t to c) at position -11 (t-11c) relative to the start codon of that reduces the expression of a monooxygenase (EthA) that activates ethionamide. (In this article, nucleotide changes are lowercase and amino acid substitutions are uppercase.) Using a flow cytometry-based reporter assay, we show that the reduced transcription of is not due to transcriptional repression by Clinical strains harboring this mutation were resistant to ethionamide. Other promoter mutations were identified in a global genomic survey of resistant strains. These results demonstrate a new mechanism of ethionamide resistance that can cause high-level resistance when it is combined with other ethionamide resistance-conferring mutations. Our study revealed many other genes which were highly up- or downregulated in XDR strains, including a toxin-antitoxin module () and tRNAs ( and ). This suggests that global transcriptional modifications could contribute to resistance or the maintenance of bacterial fitness have also occurred in XDR strains.